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Z. armatum is an economically valued crop known for its rich aroma and medicinal properties. This study identified 45 members of the SQUAMOSA-PROMOTER BINDING PROTEIN LIKE (SPL) gene family in the genome of Z. armatum. Phylogenetic and collinearity analyzes demonstrated a close relationship between ZaSPLs and ZbSPLs from B subgenomes of Zanthoxylum bungeanum. Our miRNA sequencing revealed a high degree of conservation of miR156a within Z. armatum, with the za-miR156a sequence identical to miR156-5p in Arabidopsis thaliana and Citrus sinensis. Of the 45 genes identified by ZaSPLs, 21 were targeted by za-miR156a, transient co-expression experiments in N. benthamiana demonstrated the targeting relationship between za-miR156 and ZaSPL21. Furthermore, RNA-seq and qRT-PCR analysis revealed that ZaSPL genes exhibited elevated expression levels in juvenile tissues of Z. armatum. The expression of nine representative ZaSPL genes were upregulated under polyethylene glycol (PEG) and abscisic acid (ABA). Overexpression of ZaSPL21 delayed the germination of transgenic tobacco and facilitated the flowering process in transgenic N. benthamiana. Significant up-regulation in the expression levels of flowering-related genes such as NbFT1, NbPIP2;1, NbTCP1, NbCOL1, NbGI2, NbGAI1, NbCKX2, and NbARR4 was observed in transgenic plants, suggesting that ZaSPL21 may stimulate plant flowering by regulation of these genes. Furthermore, ZaSPL21 also increased the germination speed of transgenic tobacco seeds during drought and salt stress conditions, and improved the salt tolerance of transgenic seedlings. In conclusion, our study contributes to understanding the functional analysis of the SPL gene family in Z. armatum and emphasizes the crucial role of ZaSPL21 in improving tolerance to salt and promoting flowering. The results offer potential strategies for the further utilization of these genes to improve the salt tolerance of Z. armatum., Competing Interests: Declarations. Conflict of interest: The authors have no relevant financial or non-financial interests to disclose., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Dried roots of Zanthoxylum nitidum (2n=2x=70, family Rutaceae) was referred as "Liang-Mian-Zhen" in Chinese, acting as a valuable species due to its notable pharmacological activities. Herein, we combined PacBio HiFi data together with Hi-C mapping technology to construct a chromosome-scale reference genome assembly for Z. nitidum. The assembly reached a length of 2.24 Gb, successfully anchoring 99.31% of sequences onto 35 pseudo-chromosomes. Among these, 26 chromosomes achieved telomere-to-telomere assembly, and 11 chromosomes were gap-free. The contigs N50 and scaffolds N50 reached 57.61 Mb and 78.00 Mb, respectively. Transposable elements comprised 81.44% of the Z. nitidum genome, and over 78% of them were long-terminal repeat retrotransposon elements. Furthermore, 32,737 protein-coding genes were identified and 99.38% of all were functionally annotated. The completeness of the genome assembly and final gene sets reached 97.83% and 96.47% based on Benchmarking Universal Single-Copy Orthologs (BUSCO), respectively. Taken together, our results provided a high-quality chromosome-level assembly of Z. nitidum genome and will be a valuable resource that will facilitate breeding varieties with higher alkaloids content., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Ethylene regulates fruit ripening, and in Zanthoxylum bungeanum, fruit color deepened with increasing of ethylene during fruit ripening. However, the molecular mechanism of this physiological process was still unclear. In this study, through the combined analysis of transcriptome and metabolome, it was found that ethylene release was consistent with anthocyanin synthesis, and ethylene response factors (ERFs) were significantly related to anthocyanin biosynthesis during the fruit ripening of Z. bungeanum. Ethylene treatment significantly induced fruit coloration and promoted anthocyanin synthesis and the expression of ZbERF3. Furthermore, Yeast one-hybrid assays and Luciferase reporter assays demonstrated that ZbERF3 directly bound to the promoter of ZbMYB17 and transcriptionally activated its expression. What's more, it was demonstrated that ZbMYB17 directly bound to the promoter of ZbANS, promoting anthocyanin biosynthesis. Overall, this study revealed the mechanism of ERF and MYB synergistically regulating the coloration of Z. bungeanum fruit., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Many studies have shown that seed traits, which are among the most important plant traits, can be inherited stably, a finding which is of great value for the improvement of seed germination, seed propagation, seedling establishment, plant breeding, and ecological restoration. The differences in phenotype and nutritional traits and their interactions in Zanthoxylum planispinum var. dingtanensis were ascertained, and the nutrient input rule and the strategy of resource balancing were analyzed in order to provide a scientific basis for the screening of improved variants of the test plant., Results: The nutrient distribution with in the tissues of Z. planispinum var. dingtanensis fruit was that the pericarp had adequate concentrations of N and P concentrations and the seed was also sufficient in P, but low in N concentration. Inorganic nutrients were particularly invested in the pericarp, while organic nutrients are more likely to be stored in the seed. In the economic spectrum of seed traits, the large leaf Zanthoxylum variant represented the low-investment economic type, the tufted leaf Zanthoxylum variant represented the high-investment luxury type, and the safflower Zanthoxylum and acutifoliate leaf Zanthoxylum variants represented transitional types., Conclusions: Inorganic nutrients were more invested in the pericarp to produce secondary metabolites, while organic nutrients are more likely to be stored in the seed to ensure seed germination and seedling establishment in order to achieve inheritance. The variants of Z. planispinum var. dingtanensis differ in terms of resource allocation and balance, which could be further exploited through combining characters in breeding programs., Competing Interests: Declarations Ethics approval and consent to participate This study was conducted in accordance with the relevant guidelines and legislation of the People’s Republic of China and international authorities. Permission for collection of the four variants of Z. planispinum var. dingtanensis in the experiments was obtained from Zhenfeng County, Qianxinan Prefecture, Guizhou Province, China. The Z. planispinum var. dingtanensis used in this study was identified by Changsheng Wei, Senior Engineer of Zhengfeng County Forestry, and voucher specimens are preserved in the Zhenfeng County Ding Tan Jiao Ye Co., Ltd (voucher ID: 991005, 992003, 221007, and 222005). Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Waterlogging stress negatively affects plant growth and survival. However, the ability of Zanthoxylum armatum, a valuable tree species, to tolerate and adapt to waterlogging stress remains poorly understood. Here we report how alcohol dehydrogenase 1 (ZaADH1) confers waterlogging stress tolerance in Z. armatum. ZaADH1 expression was induced after waterlogging treatment. ZaADH1 overexpression increased waterlogging stress by modulating the metabolite levels of the ADH enzyme, soluble sugar, and trehalose, promoting glycolysis and carbohydrate metabolism. The overexpression of ZaADH1 in Arabidopsis thaliana increased the total plant area and chlorophyll content, thereby increasing resistance to waterlogging stress. Physiological and overexpression transcriptome analyses in A. thaliana indicated that ZaADH1 overexpressing lines generated more carbohydrates to meet energy demands, employing a "static" strategy to increase tolerance to waterlogging stress, which confirms the conservation of the ADH1 response to waterlogging stress and represents a potential crucial measure for improving waterlogging tolerance in Z. armatum., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Masson SAS.)

Zanthoxylum is a versatile economic tree species utilized for its spice, seasoning, oil, medicinal, and industrial raw material applications, and it has a lengthy history of cultivation and domestication in China. This has led to the development of numerous cultivars. However, the phenomenon of mixed cultivars and confusing names has significantly obstructed the effective utilization of Zanthoxylum resources and industrial development. Consequently, conducting genetic diversity studies and cultivar identification on Zanthoxylum are crucial. This research analyzed the genetic traits of 80 Zanthoxylum cultivars using simple sequence repeat (SSR) and inter-Primer Binding Site (iPBS) molecular markers, leading to the creation of a DNA fingerprint. This study identified 206 and 127 alleles with 32 SSR markers and 10 iPBS markers, respectively, yielding an average of 6.4 and 12.7 alleles (Na) per marker. The average polymorphism information content (PIC) for the SSR and iPBS markers was 0.710 and 0.281, respectively. The genetic similarity coefficients for the 80 Zanthoxylum accessions ranged from 0.0947 to 0.9868 and from 0.2206 to 1.0000, with mean values of 0.3864 and 0.5215, respectively, indicating substantial genetic diversity. Cluster analysis, corroborated by principal coordinate analysis (PCoA), categorized these accessions into three primary groups. Analysis of the genetic differentiation among the three Zanthoxylum (Z. bungeanum, Z. armatum, and Z. piperitum) populations using SSR markers revealed a mean genetic differentiation coefficient (Fst) of 0.335 and a gene flow (Nm) of 0.629, suggesting significant genetic divergence among the populations. Molecular variance analysis (AMOVA) indicated that 65% of the genetic variation occurred within individuals, while 35% occurred among populations. Bayesian model-based analysis of population genetic structure divided all materials into two groups. The combined PI and PIsibs value of the 32 SSR markers were 4.265 × 10 - 27 and 1.282 × 10 - 11 , respectively, showing strong fingerprinting power. DNA fingerprints of the 80 cultivars were established using eight pairs of SSR primers, each assigned a unique numerical code. In summary, while both markers were effective at assessing the genetic diversity and relationships of Zanthoxylum species, SSR markers demonstrated superior polymorphism and cultivar discrimination compared to iPBS markers. These findings offer a scientific foundation for the conservation and sustainable use of Zanthoxylum species., (© 2024. The Author(s).)

Response Regulators (RRs) are crucial regulators in plant development and stress responses, comprising A-type, B-type, C-type, and pseudo-RR subfamilies. However, previous studies have often focused on specific subfamilies, which restricts our understanding of the complete RR gene family. In this study, we conducted a comprehensive analysis of 63 RR members from Zanthoxylum armatum, using phylogenetic relationships, motif composition, cis-acting elements, gene duplication and collinearity analyses. Segmental repeats among ZaRR genes enhanced the various environmental adaptabilities of Z. armatum, and the B-type ZaRR exhibited significant collinearity with the RRs in P. trichocarpa and C. sinensis. Cis-element analysis indicated ZaRRs play a significant role in abiotic stress and phytohormone pathways, particularly in light, drought, cold, abscisic acid (ABA) and salicylic acid (SA) responses. Abundant Ethylene Response Factor (ERF) and reproduction-associated binding sites in ZaRR promoters suggested their roles in stress and reproductive processes. A-type ZaRRs were implicated in plant vegetative and reproductive growth, whereas B-type ZaRRs contributed to both growth and stress responses. C-type ZaRRs were associated with plant reproductive growth, whereas pseudo-RRs may function in plant stress responses, such as water logging, cold, and response to ethylene (ETH), SA, and jasmonic acid (JA). Ectopic expression of ZaRR24, a C-type RR, inhibits growth, induces early flowering, and shortens fruit length in Arabidopsis. ZaRR24 overexpression also affected the expression of A- and B-type RRs, as well as floral meristem and organ identity genes. These findings establish a solid and comprehensive foundation for RR gene research in Z. armatum, and provide a platform for investigating signal transduction in other woody plants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier Masson SAS. All rights reserved.)

Anthocyanin (ACN)-derived pigmentation in the red Zanthoxylum bungeanum peel is an essential commercial trait. Therefore, exploring the metabolic regulatory networks involved in peel ACN levels in this species is crucial for improving its quality. However, its underlying transcriptional regulatory mechanisms are still unknown. This transcriptomic and bioinformatics study not only discovered a new TF ( ZbMYB111 ) as a potential regulator for ACN biosynthesis in Z. bungeanum peel, but also deciphered the underlying molecular mechanisms of ACN biosynthesis. Overexpression of ZbMYB111 and flavonoid 3-O-glucosyltransferase ( ZbUFGT ) induced ACN accumulation in both Z. bungeanum peels and callus along with Arabidopsis thaliana and tobacco flowers, whereas their silencing impaired ACN biosynthesis. Therefore, the dual-luciferase reporter, yeast-one-hybrid, and electrophoretic mobility shift assays showed that ZbMYB111 directly interacted with the ZbUFGT promoter to activate its expression. This diverted the secondary metabolism toward the ACN pathway, thereby promoting ACN accumulation.

Main Conclusion: In this study, six ZaBZRs were identified in Zanthoxylum armatum DC, and all the ZaBZRs were upregulated by abscisic acid (ABA) and drought. Overexpression of ZaBZR1 enhanced the drought tolerance of transgenic Nicotiana benthamian. Brassinosteroids (BRs) are a pivotal class of sterol hormones in plants that play a crucial role in plant growth and development. BZR (brassinazole resistant) is a crucial transcription factor in the signal transduction pathway of BRs. However, the BZR gene family members have not yet been identified in Zanthoxylum armatum DC. In this study, six members of the ZaBZR family were identified by bioinformatic methods. All six ZaBZRs exhibited multiple phosphorylation sites. Phylogenetic and collinearity analyses revealed a closest relationship between ZaBZRs and ZbBZRs located on the B subgenomes. Expression analysis revealed tissue-specific expression patterns of ZaBZRs in Z. armatum, and their promoter regions contained cis-acting elements associated with hormone response and stress induction. Additionally, all six ZaBZRs showed upregulation upon treatment after abscisic acid (ABA) and polyethylene glycol (PEG), indicating their participation in drought response. Subsequently, we conducted an extensive investigation of ZaBZR1. ZaBZR1 showed the highest expression in the root, followed by the stem and terminal bud. Subcellular localization analysis revealed that ZaBZR1 is present in the cytoplasm and nucleus. Overexpression of ZaBZR1 in transgenic Nicotiana benthamiana improved seed germination rate and root growth under drought conditions, reducing water loss rates compared to wild-type plants. Furthermore, ZaBZR1 increased proline content (PRO) and decreased malondialdehyde content (MDA), indicating improved tolerance to drought-induced oxidative stress. The transgenic plants also showed a reduced accumulation of reactive oxygen species. Importantly, ZaBZR1 up-regulated the expression of drought-related genes such as NbP5CS1, NbDREB2A, and NbWRKY44. These findings highlight the potential of ZaBZR1 as a candidate gene for enhancing drought resistance in transgenic N. benthamiana and provide insight into the function of ZaBZRs in Z. armatum., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: AP2/ERF is a large family of plant transcription factor proteins that play essential roles in signal transduction, plant growth and development, and responses to various stresses. The AP2/ERF family has been identified and verified by functional analysis in various plants, but so far there has been no comprehensive study of these factors in Chinese prickly ash. Phylogenetic, motif, and functional analyses combined with transcriptome analysis of Chinese prickly ash fruits at different developmental stages (30, 60, and 90 days after anthesis) were conducted in this study., Results: The analysis identified 146 ZbAP2/ERF genes that could be classified into 15 subgroups. The motif analysis revealed the presence of different motifs or elements in each group that may explain the functional differences between the groups. ZbERF13.2, ZbRAP2-12, and ZbERF2.1 showed high levels of expression in the early stages of fruit development. ZbRAP2-4, and ZbERF3.1 were significantly expressed at the fruit coloring stage (R2 and G2). ZbERF16 were significantly expressed at fruit ripening and expression level increased as the fruit continued to develop. Relative gene expression levels of 6 representative ZbAP2/ERFs assessed by RT-qPCR agreed with transcriptome analysis results., Conclusions: These genes identified by screening can be used as candidate genes that affect fruit development. The results of the analysis can help guide future genetic improvement of Chinese prickly ash and enrich our understanding of AP2/ERF transcription factors and their regulatory functions in plants., (© 2024. The Author(s).)

Apomixis is a common reproductive characteristic of Zanthoxylum plants, and RWP-RKs are plant-specific transcription factors known to regulate embryonic development. However, the genome-wide analysis and function prediction of RWP-RK family genes in Z. armatum are unclear. In this study, 36 ZaRWP-RK transcription factors were identified in the genome of Z. armatum , among which 15 genes belonged to the RKD subfamily and 21 belonged to the NLP subfamily. Duplication events of ZaRWP-RK genes were mainly segmental duplication, and synteny analysis revealed a close phylogenetic relationship between Z. armatum and Arabidopsis . The analysis of cis-elements indicated that ZaRWP-RK genes may be involved in the regulation of the embryonic development of Z. armatum by responding to plant hormones such as abscisic acid, auxin, and gibberellin. Results of a real-time PCR showed that the expression levels of most ZaRWP-RK genes were significantly increased from flowers to young fruits. Protein-protein interaction network analysis further revealed the potential roles of the ZaRWP-RK proteins in apomixis. Collectively, this study is expected to improve our understanding of ZaRWP-RK transcription factors and provide a theoretical basis for future investigations into the ZaRWP-RK genes and their regulatory mechanisms in the apomixis process of Z. armatum .

Terpenoids are the main active components in the Zanthoxylum armatum leaves, which have extensive medicinal value. The Z. armatum leaf is the main by-product in the Z. armatum industry. However, the transcription factors involved in the biosynthesis of terpenoids are rarely reported. This study was performed to identify and classify the APETALA2/ethylene-responsive factor (AP2/ERF) gene family of Z. armatum. The chromosome distribution, gene structure, conserved motifs, and cis-acting elements of the promoter of the species were also comprehensively analyzed. A total of 214 ZaAP2/ERFs were identified. From the obtained transcriptome and terpenoid content data, four candidate ZaAP2/ERFs involved in the biosynthesis of terpenoids were selected via correlation and weighted gene co-expression network analysis. A phylogenetic tree was constructed using 13 AP2/ERFs related to the biosynthesis of terpenoids in other plants. ZaERF063 and ZaERF166 showed close evolutionary relationships with the ERFs in other plant species and shared a high AP2-domain sequence similarity with the two closest AP2/ERF proteins, namelySmERF8 from Salvia miltiorrhiza and AaERF4 from Artemisia annua. Further investigation into the effects of methyl jasmonate (MeJA) treatment on the content of terpenoids in Z. armatum leaves revealed that MeJA significantly induced the upregulation of ZaERF166 and led to a significant increase in the terpenoids content in Z. armatum leaves, indicating that ZaERF166 might be involved in the accumulation of terpenoids of Z. armatum. Results will be beneficial for the functional characterization of AP2/ERFs in Z. armatum and establishment of the theoretical foundation to increase the production of terpenoids via the manipulation of the regulatory elements and strengthen the development and utilization of Z. armatum leaves., (© 2023 The Authors. The Plant Genome published by Wiley Periodicals LLC on behalf of Crop Science Society of America.)

Zanthoxylum nitidum (Roxb.) is a commonly used traditional Chinese medicine. However, the collection and protection of wild germplasm resources of Z. nitidum are still insufficient, and there is limited research on its genetic diversity and fingerprint. In the present study, 15 simple sequence repeat (SSR) markers were developed by genotyping based on multiplexed shotgun sequencing. The genetic diversity of 51 populations (142 individuals) of Z. nitidum was evaluated using these 15 SSRs. A total of 245 alleles (Na) were detected, with an average value of 16.333, and the average polymorphism information content was 0.756. The genetic distance among 51 populations was 0.164~1.000, with an average of 0.659. Analysis of molecular variance showed low genetic differentiation (40%) and high genetic differentiation (60%) between populations and individuals, respectively. The genetic differentiation coefficient (Fst) of the population was 0.338, indicating that 66.2% of the genetic variation occurred within the population, and the gene flow (Nm) was 0.636, demonstrating that the gene exchange between populations was low. Clustering analysis revealed that the genetic similarity coefficient was 0.30, dividing the 51 populations into 4 groups of 2, 17, 3, and 29 populations. There was no specific relationship between geographical location differences and genetic distance. The genetic diversity level of Z. nitidum is relatively high, and our results provide a theoretical basis for the rapid identification of Z. nitidum germplasm resources and variety selection., (© 2023. The Author(s).)

Chinese pepper rust is a live parasitic fungal disease caused by Coleosporium zanthoxyli , which seriously affects the cultivation and industrial development of Z. armatum . Cultivating and planting resistant cultivars is considered the most economical and environmentally friendly strategy to control this disease. Therefore, the mining of excellent genes for rust resistance and the analysis of the mechanism of rust resistance are the key strategies to achieve the targeted breeding of rust resistance. However, there is no relevant report on pepper rust resistance at present. The aim of the present study was to further explore the resistance mechanism of pepper by screening the rust-resistant germplasm resources in the early stage. Combined with the analysis of plant pathology, transcriptomics, and metabolomics, we found that compared with susceptible cultivar TJ, resistant cultivar YK had 2752 differentially expressed genes (DEGs, 1253 up-, and 1499 downregulated) and 321 differentially accumulated metabolites (DAMs, 133 up- and 188 down-accumulated) after pathogen infection. And the genes and metabolites related to phenylpropanoid metabolism were highly enriched in resistant varieties, which indicated that phenylpropanoid metabolism might mediate the resistance of Z. armatum . This finding was further confirmed by a real-time quantitative polymerase chain reaction analysis, which revealed that the expression levels of core genes involved in phenylpropane metabolism in disease-resistant varieties were high. In addition, the difference in flavonoid and MeJA contents in the leaves between resistant and susceptible varieties further supported the conclusion that the flavonoid pathway and methyl jasmonate may be involved in the formation of Chinese pepper resistance. Our research results not only help to better understand the resistance mechanism of Z. armatum rust but also contribute to the breeding and utilization of resistant varieties.

Zanthoxylum bungeanum is an important condiment with high economic value and its peel color is one of the main quality indexes. However, the key pigment compounds and related genes are still unclear affecting the quality control of the plants. In this study, the contents of four types of pigments were measured in Z. bungeanum and flavonoids were identified as the most important pigments. Based on the targeted flavonoid metabolomics of Z. bungeanum peels, 14 key pigment compounds were screened out from 152 flavonoids, among which cyanidin-3-O-rutinoside and cyanidin-3-O-glucoside were the most critical compounds for peel color. They were further verified to be present in nine varieties of Z. bungeanum by HPLC fingerprints. The 14 compounds were all associated with flavonoid and anthocyanin biosynthesis pathways and the 39 differentially expressed genes related to these pathways were annotated and screened based on transcriptomics. The genes ZbDFR, ZbANS, and ZbUFGT were identified as three key genes for anthocyanin synthesis in Z. bungeanum peels. Further qRT-PCR results confirmed the reliability of transcriptomics and the accuracy of gene screening. Subsequent protein induced expression demonstrated that ZbANS and ZbUFGT were expressed after 12 h induced by IPTG while ZbDFR was expressed after 15 h. Further transient and stable transformation analysis confirmed that both anthocyanin content and the expression of ZbDFR were significantly increased in overexpression Z. bungeanum leaves and Nicotiana benthamiana. The functional effect of stable transformation of ZbDFR was more significant than that of transient transformation with a 7.67-fold/1.49-fold difference in total anthocyanin content and a 42.37-fold/12.32-fold difference in the expression of ZbDFR. This study provides new insights into the chemical composition and the molecular mechanisms of Z. bungeanum peel color and lays an effective foundation for the color quality control, multi-purpose utilization of Z. bungeanum and the creation of new germplasm., (© 2023 Scandinavian Plant Physiology Society.)

Tana (Zanthoxylum ailanthoides), a perennial deciduous species in the Rutaceae family, possesses leaves with a unique fragrance that indigenous peoples incorporate into their traditional cuisine. In Kalibuan, the cultivated tana trees were pruned repeatedly to maintain a shorter height, which led to the growth of new leaves that were spicier and pricklier. Tana leaves contain a range of volatile terpenoids, and the pungent aroma may arise from the presence of monoterpenoids. To gain insight into the biosynthetic pathway, five candidate monoterpene synthase genes were cloned and characterized using a purified recombinant protein assay. The main product of Za_mTPS1, Za_mTPS2, and Za_mTPS5 is sabinene, geraniol, and (E)-β-ocimene, respectively. The main product of Za_mTPS3 and Za_mTPS4 is linalool. Real-time PCR analysis revealed that Za_mTPS1 and Za_mTPS5 are expressed at higher levels in prickly leaves of cultivated tana, suggesting that they may contribute to the distinctive aroma of this plant., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Masson SAS.)

Zanthoxylum armatum is a dioecious prickly plant which developed apomictic reproduction. The increases in male flowers and prickle density in female plants lead to low yield and picking efficiency. However, little is known concerning the mechanisms of floral development and prickle formation. NAC is a well-known transcription factor that participates in multiple aspects of plant growth and development. Herein, we characterize the functions and regulatory mechanisms of candidate NACs controlling both traits in Z. armatum. A total of 159 ZaNACs were identified, and 16 of these were male-biased, represented by the NAP subfamily members ZaNAC93 and ZaNAC34, orthologs of AtNAC025 and AtNARS1/NAC2 respectively. Overexpression of ZaNAC93 in tomato led to modifications in flower and fruit development, including earlier flowering, increased numbers of lateral shoots and flowers, accelerated plant senescence, and reduced size and weight of fruits and seeds. In addition, the trichome density in leaves and inflorescences was dramatically reduced in ZaNAC93-OX lines. Overexpression of ZaNAC93 resulted in the up-/downregulation of genes associated with GA, ABA and JA signaling pathways, such as GAI, PYL and JAZ, as well as several TFs, including bZIP2, AGL11, FBP24 and MYB52. Yeast two-hybrid analysis revealed that ZaNAC93 protein could interact with AP1, GAI, bZIP2 and AGL11 in Z. armatum, which might contribute to floral induction, fruit growth, and trichome initiation. This work provides new insights into the molecular mechanisms of ZaNAC93 in reproductive development and prickle formation in Z. armatum., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Green prickly ash ( Zanthoxylum armatum ) has edible and medicinal value and is an economically significant plant in many countries. Z. armatum has many cultivars and varieties with similar phenotypes that are difficult to distinguish via traditional methods. In this study, we utilized oligo-FISH to distinguish five varieties and cultivars of Z. armatum on the basis of three oligonucleotide probes of 5S rDNA, (AG 3 T 3 ) 3 , and (GAA) 6 . Karyotype analysis of the five varieties and cultivars of Z. armatum showed that the Z. armatum 'Tengjiao' karyotype formula was 2 n = 2 x = 98 m with karyotype type 1C and an arm ratio of 4.3237, including two pairs of 5S rDNA signals and five pairs of (GAA) 6 signals. The karyotype formula of Z. armatum 'Youkangtengjiao' was 2 n = 2 x = 128 m + 8 sm with karyotype type 2B and an arm ratio of 3.5336, including three pairs of 5S rDNA signals and 17 pairs of (GAA) 6 signals. The karyotype formula of Z. armatum var. novemfolius was 2 n = 2 x = 134 m + 2 sm with karyotype type 1C and an arm ratio of 5.5224, including two pairs of 5S rDNA signals and eight pairs of (GAA) 6 signals. The karyotype formula of Z. armatum 'YT-03' was 2 n = 2 x = 2 M + 128 m + 4 sm + 2 st with karyotype type 2C and an arm ratio of 4.1829, including three pairs of 5S rDNA signals and nine pairs of (GAA) 6 signals. The karyotype formula of Z. armatum 'YT-06' was 2 n = 2 x = 126 m + 10 sm with cytotype 2B and an arm ratio of 3.3011, including three pairs of 5S rDNA signals and two pairs of (GAA) 6 signals. The five varieties and cultivars of Z. armatum had (AG 3 T 3 ) 3 signals on all chromosomes. The chromosomal symmetry of Z. armatum 'Tengjiao' was high, whereas the chromosomal symmetry of Z. armatum 'YT-03' was low, with the karyotypes of the five materials showing a trend toward polyploid evolution. The phylogenetic relationship between Z. armatum 'Tengjiao' and Z. armatum var. novemfolius was the closest, while that between Z. armatum 'YT-03' and Z. armatum 'YT-06' was closer than with Z. armatum 'Youkangtengjiao' according to oligo-FISH. The results provided a karyotype profile and a physical map that contributes to the distinction of varieties and cultivars of Z. armatum and provides strategies for distinguishing other cultivated species.

Zanthoxylum armatum is an evergreen plant with high economical and medicinal values. The presence of prickles on stems and leaves is undesirable for them make picking difficult. To date, little is known of prickle formation in Z. armatum. Herein, the morphological and molecular features of prickle initiation in prickless (WC) and three types of prickly Z. armatum were characterized. Compared to WC, the levels of cytokinin and auxin were increased, while GA and JA declined in prickly Z. armatum. Transcriptome analysis identified 6258 differentially expressed genes (DEGs) between prickless and prickly Z. armatum. Among them, several DEGs related to hormone metabolism and signaling, including LOG7, CKX3, AHK1, three DELLAs, six JAZs and TIR1, were candidate genes involved in prickle formation. Transcription factors associated with prickle formation were screened, including MYB6-1/MYB6-2, WER, GL3-2, SPL4/5, SOC1, and SCL32. Of them, MYB6-1 and WER might negatively regulate prickles initiation via interacting with GL3-2. Additionally, the histone acetylation and DNA methylation levels, the transcripts of histone acetyltransferase/deacetylase and DNA methyltransferases showed significant differences between prickless and prickly plants, indicating their involvements in prickle initiation. These findings illustrate the regulation of prickle formation might be mediated by phytohormones (especially cytokinin), transcription factors and epigenetic modifications in Z. armatum., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Zanthoxylum nitidum (Roxb.) DC. ( Z. nitidum ) is a type of Chinese Dao-di herb, also called Liangmianzhen, which is widely used to treat arthralgia, rheumatic arthralgia, and stomach pain. However, genomic resources for Z. nitidum are still scarce. This study provides transcriptomic resources for Z. nitidum by applying single-molecule real-time (SMRT) sequencing technology. In total, 456,109 circular consensus sequencing (CCS) reads were generated with a mean length of 2,216 bp from Z. nitidum roots, old stems, young branches, leaves, flowers, and fruits. Of these total reads, 353,932 were full-length nonchimeric (FLNC) reads with an average length of 1,996 bp. A total of 16,163 transcripts with a mean length of 1,171 bp were acquired. Of these transcripts, 14,231 (88%) were successfully annotated using public databases. Across all the 16,163 transcripts, we identified 6,255 long non-coding RNAs (lncRNAs) and 22,780 simple sequence repeats (SSRs). Furthermore, 3,482 transcription factors were identified. Among the SSR loci, 1-3 nucleotide repeats were dominant, occupying 99.36% of the total SSR loci, with mono-, di-, and tri-nucleotide repeats accounting for 61.80%, 19.89%, and 5.02% of the total SSR loci, respectively. A total of 36 out of 100 randomly selected primer pairs were verified to be positive, 20 of which showed polymorphism. These findings enrich the genetic resources available for facilitating future studies and research on relevant topics such as population genetics in Z. nitidum ., Competing Interests: Zhu Yanxia, Huang Yanfen, Wei Kunhua and Jiang Jianping are all scientific researchers of Guangxi Medical Botanical Garden. Junnan Yu is employed by ChongQing Jinzhi Quality Certification Co., LTD. The authors declare that they have no competing interests., (© 2023 Zhu et al.)

Zanthoxylum armatum is an important cash crop for medicinal and food purposes in Asia. However, its stems and leaves are covered with a large number of prickles, which cause many problems in the production process. The homeodomain leucine zipper (HD-ZIP) gene family is a class of transcription factors unique to plants that play an important role in biological processes such as morphogenesis, signal transduction, and secondary metabolite synthesis. However, little is known about HD-ZIP gene information that may be involved in prickle development of Z. armatum. Here, we identified 76 ZaHDZ genes from the Z. armatum genome and classified them into four subfamilies (I-IV) based on phylogenetic analysis, a classification further supported by gene structure and conserved motif analysis. Seventy-six ZaHDZ genes were unevenly distributed on chromosomes. Evolutionary analysis revealed that the expansion of ZaHDZ genes mainly were due to whole-genome duplication (WGD) or segmental duplication, and they experienced strong purifying selection pressure in the process of evolution. A total of 47 cis-elements were identified in the promoter region of ZaHDZ genes. Quantitative real-time polymerase chain reaction analysis was performed on subfamily IV ZaHDZ gene expression levels in five tissues and under four hormone treatments. Finally, ZaHDZ16 was predicted to be the candidate gene most likely to be involved in prickle development of Z. armatum. These results contribute to a better understanding of the characteristics of HD-ZIP gene family and lay a foundation for further study on the function of genes related to prickle development of Z. armatum., (© 2023 The Authors. The Plant Genome published by Wiley Periodicals LLC on behalf of Crop Science Society of America.)

Accumulation of anthocyanins largely determines the fruit color, and dihydroflavonol 4-reductase (DFR) is a key enzyme involved in the formation of anthocyanins. However, the catalytic and regulatory mechanisms of DFR are unclear. In this study, the gene encoding DFR from Zanthoxylum bungeanum Maxim. was cloned and ZbDFR was analyzed in detail. The ZbDFR accepted dihydrokaempferol, dihydroquercetin and dihydromyricetin as substrates. Flavonols such as myricetin, quercetin and kaempferol significantly inhibited the activity of ZbDFR, while quercitrin and isoquercitrin slightly increased the activity. Quercetin was a competitive inhibitor at low concentrations, and it had a combined effect of competitive and noncompetitive inhibition at high concentrations, which was consistent with ZbDFR having two inhibitor binding sites. In addition, the content of different types of flavonoids in Z. bungeanum peel at green, semi-red and red stage was analyzed, and the in vivo results could be explained by the regulation of ZbDFR activity in vitro. Site-directed mutagenesis combined with enzyme activity experiments showed that Ser128, Tyr163, Phe164 and Lys167 are the key catalytic amino acid residues. The Ser128, Tyr163 and Lys167 were crucial for the hydrogen transfer reaction, and mutation of these amino acids resulted in the loss of all or most of the activity. Phe164 was found to be important for the regulation of ZbDFR by flavonols. Accordingly, ZbDFR is a node at which flavonoids regulate the synthesis of anthocyanins and proanthocyanins., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Heat stress has adverse effects on the growth and reproduction of plants. Zygophyllum xanthoxylum, a typical xerophyte, is a dominant species in the desert where summer temperatures are around 40 °C. However, the mechanism underlying the thermotolerance of Z. xanthoxylum remained unclear., Results: Here, we characterized the acclimation of Z. xanthoxylum to heat using a combination of physiological measurements and transcriptional profiles under treatments at 40 °C and 45 °C, respectively. Strikingly, moderate high temperature (40 °C) led to an increase in photosynthetic capacity and superior plant performance, whereas severe high temperature (45 °C) was accompanied by reduced photosynthetic capacity and inhibited growth. Transcriptome profiling indicated that the differentially expressed genes (DEGs) were related to transcription factor activity, protein folding and photosynthesis under heat conditions. Furthermore, numerous genes encoding heat transcription shock factors (HSFs) and heat shock proteins (HSPs) were significantly up-regulated under heat treatments, which were correlated with thermotolerance of Z. xanthoxylum. Interestingly, the up-regulation of PSI and PSII genes and the down-regulation of chlorophyll catabolism genes likely contribute to improving plant performance of Z. xanthoxylum under moderate high temperature., Conclusions: We identified key genes associated with of thermotolerance and growth in Z. xanthoxylum, which provide significant insights into the regulatory mechanisms of thermotolerance and growth regulation in Z. xanthoxylum under high temperature conditions., (© 2023. The Author(s).)

Zanthoxylum armatum and Zanthoxylum bungeanum, known as 'Chinese pepper', are distinguished by their extraordinary complex genomes, phenotypic innovation of adaptive evolution and species-special metabolites. Here, we report reference-grade genomes of Z. armatum and Z. bungeanum. Using high coverage sequence data and comprehensive assembly strategies, we derived 66 pseudochromosomes comprising 33 homologous phased groups of two subgenomes, including autotetraploid Z. armatum. The genomic rearrangements and two whole-genome duplications created large (~4.5 Gb) complex genomes with a high ratio of repetitive sequences (>82%) and high chromosome number (2n = 4x = 132). Further analysis of the high-quality genomes shed lights on the genomic basis of involutional reproduction, allomones biosynthesis and adaptive evolution in Chinese pepper, revealing a high consistent relationship between genomic evolution, environmental factors and phenotypic innovation. Our study provides genomic resources and new insights for investigating diversification and phenotypic innovation in Chinese pepper, with broader implications for the protection of plants under severe environmental changes., (© 2022 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Zanthoxylum armatum DC. is a plant with many medicinal values which is extensively used in traditional system of medicine for curing various diseases and ailments, including cancer. The aim of the present study is to identify Zanthoxylum armatum collected from different parts of Manipur, India, at molecular level. Molecular markers like internal transcribed spacer (ITS) region and other DNA barcoding genes such as matK, rbcL, psbA-trnH and trnL-trnF were targeted to find out the most suitable DNA barcode for identifying this species. Sequences obtained using the five primer pairs-ITS An5 and ITS An4, matK-413f-1 and matK-1227r-1, rbcL-1F and rbcL-724R, psbA-F and trnH-R and trnL-F and trnF-R were submitted to GenBank, NCBI. Amongst the five DNA barcoding targets, one nuclear and four chloroplast genes were successfully amplified by PCR (100%) and sequencing (100%) in all the eight plant samples. Sequence similarity of total ITS region (620 bp) when compared to the reference sequence were found to be between 98.55 and 99.68%. In our study, ITS sequence in combination with DNA barcoding sequences of rbcL, trnH-psbA and trnL-trnF was very successful in identification of Z. armatum and differentiate other species clearly in the phylogeny analysis. Our work shows ITS region to be the most suitable DNA barcode which formed a monophyletic group of the species in the phylogenetic tree analysis. The sequences of the barcoding genes of Z. armatum DC. obtained from this study adds to the already available resources which will be helpful in the future research endeavours., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Multiprotein bridging factor 1 (MBF1) is a crucial transcriptional coactivator in animals, plants, and some microorganisms, that plays a necessary role in growth development and stress tolerance. Zanthoxylum armatum is an important perennial plant for the condiments and pharmaceutical industries, whereas the potential information in the genes related to stress resistance remains poorly understood in Z. armatum.  RESULTS: Herein, six representative species were selected for use in a genome-wide investigation of the MBF1 family, including Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, Citrus sinensis, Ginkgo biloba, and Z. armatum. The results showed that the MBF1 genes could be divided into two groups: Group I contained the MBF1a and MBF1b subfamilies, and group II was independent of the MBF1c subfamily.. Most species have at least two different MBF1 genes, and MBF1c is usually an essential member. The three ZaMBF1 genes were respectively located on ZaChr26, ZaChr32, and ZaChr4 of Zanthoxylum chromosomes. The collinearity were occurred between three ZaMBF1 genes, and ZaMBF1c showed the collinearity between Z. armatum and both P. trichocarpa and C. sinensis. Moreover, many cis-elements associated with abiotic stress and phytohormone pathways were detected in the promoter regions of MBF1 of six representative species. The ERF binding sites were the most abundant targets in the sequences of the ZaMBF1 family, and some transcription factor sites related to floral differentiation were also identified in ZaMBF1c, such as MADS, LFY, Dof, and AP2. ZaMBF1a was observed to be very highly expressed in 25 different samples except in the seeds, and ZaMBF1c may be associated with the male and female floral initiation processes. In addition, expression in all the ZaMBF1 genes could be significantly induced by water-logging, cold stress, ethephon, methyl jasmonate, and salicylic acid treatments, especially in ZaMBF1c., Conclusion: The present study carried out a comprehensive bioinformatic investigation related to the MBF1 family in six representative species, and the responsiveness of ZaMBF1 genes to various abiotic stresses and phytohormone inductions was also revealed. This work not only lays a solid foundation to uncover the biological roles of the ZaMBF1 family in Z. armatum, but also provides some broad references for conducting the MBF1 research in other plants., (© 2022. The Author(s).)

Growth-regulating factors (GRFs) are plant-specific transcription factors that play an important role in plant growth and development. In this study, fifteen GRF gene members containing QLQ and WRC domains were identified in Zanthoxylum armatum . Phylogenetic and collinearity analysis showed that ZaGRFs were closely related to CsGRFs and AtGRFs, and distantly related to OsGRFs. There are a large number of cis-acting elements related to hormone response and stress induction in the GRF gene promoter region of Z. armatum . Tissue-specific expression analysis showed that except for ZaGRF7 , all the ZaGRFs were highly expressed in young parts with active growth and development, including terminal buds, seeds, and young flowers, suggesting their key roles in Z. armatum growth and development. Eight ZaGRFs were selected to investigate the transcriptional response to auxin, gibberellin and drought treatments. A total of six ZaGRFs in the NAA treatment, four ZaGRFs in the GA 3 treatment, and six ZaGRFs in the PEG treatment were induced and significantly up-regulated. Overexpression of ZaGRF6 increased branching and chlorophyll content and delayed senescence of transgenic Nicotiana benthamiana . ZaGRF6 increased the expression of CRF2 and suppressed the expression of ARR4 and CKX1 , indicating that ZaGRF6 is involved in cytokinin metabolism and signal transduction. These research results lay a foundation for further analysis of the GRF gene function of Z. armatum and provide candidate genes for growth, development, and stress resistance breeding of Z. armatum .

Background: Flavonoids have strong free radical scavenging and antioxidant capacity. The high abundance of flavonoids in Chinese prickly ash peels have many benefits to human health. In this study, 'Hancheng Dahongpao', a main cultivar, was taken as materials to investigate the flavonoids biosynthesis mechanism of Zanthoxylum bungeanum Maxim at three key development stages by integration of metabolomics and transcriptomics analysis., Results: A total of 19 differentially accumulated metabolites were identified, the key flavonoids compounds were kaempferol, quercetin and their glycoside derivatives, and two major anthocyanins (peonidin O-hexoside and peonidin 3-O-glucoside). 5 gene networks/modules including 15 important candidate genes were identified, which was highly correlated with flavonoids. Among these genes, ZM-163828 and ZM-184209 were strongly correlated with kaempferol and quercetin, and ZM-125833 and ZM-97481 were controlled the anthocyanins biosynthesis. Moreover, it was shown that MYB-ZM1, MYB-ZM3, MYB-ZM5, MYB-ZM6 and MYB-ZM7 coordinately controlled flavonoids accumulation through regulating the structural genes., Conclusions: Generally, this study systematically revealed the flavonoids metabolic pathways and candidate genes involved in flavonoids biosynthesis and laid a foundation for the potential targets for the breeding of new valuable Chinese prickly ash cultivars., (© 2022. The Author(s).)

Zanthoxylum armatum DC. is an important economic tree species. Prickle is a type of trichome with special morphology, and there are a lot of prickles on the leaves of Z. armatum, which seriously restricts the development of Z. armatum industry. In this study, the leaves of Z. armatum cv. Zhuye (ZY) and its budding variety 'Rongchangwuci' (WC) (A less prickly mutant variety) at different developmental stages were used as materials, and the transcriptome sequencing data were analyzed. A total of 96,931 differentially expressed genes (DEGs) were identified among the samples, among which 1560 were candidate DEGs that might be involved in hormone metabolism. The contents of JA, auxin and CK phytohormones in ZY leaves were significantly higher than those in WC leaves. Combined with weighted gene co-expression network analysis, eight genes (MYC, IAA, ARF, CRE/AHK, PP2C, ARR-A, AOS and LOX) were identified, including 25 transcripts, which might affect the metabolism of the three hormones and indirectly participate in the formation of prickles. Combining with the proteins successfully reported in other plants to regulate trichome formation, ZaMYB86, a transcription factor of R2R3 MYB family, was identified through local Blast and phylogenetic tree analysis, which might regulate prickle formation of Z. armatum. Overexpression of ZaMYB86 in mutant A. thaliana resulted in the reduction of trichomes in A. thaliana leaves, which further verified that ZaMYB86 was involved in the formation of pickles. Yeast two-hybrid results showed that ZaMYB86 interacted with ZaMYB5. Furthermore, ZaMYB5 was highly homologous to AtMYB5, a transcription factor that regulated trichomes development, in MYB DNA binding domain. Taken together, these results indicated that ZaMYB86 and ZaMYB5 act together to regulate the formation of prickles in Z. armatum. Our findings provided a new perspective for revealing the molecular mechanism of prickly formation., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

NAC (NAM, ATAF1/2, and CUC2) transcription factors (TFs) are one of the largest plant-specific TF families and play a pivotal role in adaptation to abiotic stresses. The genome-wide analysis of NAC TFs is still absent in Zanthoxylum bungeanum . Here, 109 ZbNAC proteins were identified from the Z. bungeanum genome and were classified into four groups with Arabidopsis NAC proteins. The 109 ZbNAC genes were unevenly distributed on 46 chromosomes and included 4 tandem duplication events and 17 segmental duplication events. Synteny analysis of six species pairs revealed the closely phylogenetic relationship between Z. bungeanum and C . sinensis . Twenty-four types of cis -elements were identified in the ZbNAC promoters and were classified into three types: abiotic stress, plant growth and development, and response to phytohormones. Co-expression network analysis of the ZbNAC s revealed 10 hub genes, and their expression levels were validated by real-time quantitative polymerase chain reaction (qRT-PCR). Finally, ZbNAC007 , ZbNAC0 18 , ZbNAC047 , ZbNAC072 , and ZbNAC079 were considered the pivotal NAC genes for drought tolerance in Z. bungeanum . This study represented the first genome-wide analysis of the NAC family in Z. bungeanum , improving our understanding of NAC proteins and providing useful information for molecular breeding of Z. bungeanum .

Aflatoxin B1 (AFB1) is a food-borne toxin produced by Aspergillus flavus and a few similar fungi. Natural anti-aflatoxigenic compounds are used as alternatives to chemical fungicides to prevent AFB1 accumulation. We found that a methanolic extract of the food additive Zanthoxylum bungeanum shuts down AFB1 production in A. flavus. A methanol sub-fraction (M20) showed the highest total phenolic/flavonoid content and the most potent antioxidant activity. Mass spectrometry analyses identified four flavonoids in M20: quercetin, epicatechin, kaempferol-3-O-rhamnoside, and hyperoside. The anti-aflatoxigenic potency of M20 (IC 50 : 2-4 µg/mL) was significantly higher than its anti-proliferation potency (IC 50 : 1800-1900 µg/mL). RNA-seq data indicated that M20 triggers significant transcriptional changes in 18 of 56 secondary metabolite pathways in A. flavus, including repression of the AFB1 biosynthesis pathway. Expression of aflR, the specific activator of the AFB1 pathway, was not changed by M20 treatment, suggesting that repression of the pathway is mediated by global regulators. Consistent with this, the Velvet complex, a prominent regulator of secondary metabolism and fungal development, was downregulated. Decreased expression of the conidial development regulators brlA and Medusa, genes that orchestrate redox responses, and GPCR/oxylipin-based signal transduction further suggests a broad cellular response to M20. Z. bungeanum extracts may facilitate the development of safe AFB1 control strategies., (© 2022. The Author(s).)

Sichuan pepper (Zanthoxylum armatum DC) is a popular spice and is often prescribed in traditional Chinese medicine to treat vomiting, diarrhea, ascariasis and eczema, among other conditions. Volatile oils from Z. armatum leaves contain active ingredients, with terpenoids being one of the main components. In the present study, the combination of sequencing data of Z. armatum from PacBio single molecule real time (SMRT) and Illumina RNA sequencing (RNA-Seq) platforms facilitated an understanding of the gene regulatory network of terpenoid biosynthesis in pepper leaves. The leaves of three developmental stages from two Z. armatum cultivars, 'Rongchangwuci' (WC) and 'Zhuye' (ZY), were selected as test materials to construct sequencing libraries. A total of 143,122 predictions of unique coding sequences, 105,465 simple sequence repeats, 20,145 transcription factors and 4719 long non-coding RNAs (lncRNAs) were identified, and 142,829 transcripts were successfully annotated. The occurrence of alternative splicing events was verified by reverse transcription PCR, and quantitative real-time PCR was used to confirm the expression pattern of six randomly selected lncRNAs. A total of 96,931 differentially expressed genes were filtered from different samples. According to functional annotation, a total of 560 candidate genes were involved in terpenoid synthesis, of which 526 were differentially expressed genes (DEGs). To identify the key genes involved in terpenoid biosynthesis, the module genes in different samples, including structural and transcription factors genes, were analyzed using the weighted gene co-expression network method, and the co-expression network of genes was constructed. Thirty-one terpenoids were identified by gas chromatography-mass spectrometry. The correlation between 18 compounds with significantly different contents and genes with high connectivity in the module was jointly analyzed in both cultivars, yielding 12 candidate DEGs presumably involved in the regulation of terpenoid biosynthesis. Our findings showed that full-length transcriptome SMRT and Illumina RNA-Seq can play an important role in studying organisms without reference genomes and elucidating the gene regulation of a biosynthetic pathway., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Pericarp color is an important economic characteristic of Zanthoxylum bungeanum . Anthocyanins are the main reason for the pericarp's red appearance in Z. bungeanum . In this study, through the combined analysis of the metabolome and transcriptome, HY5 , whose expression is highly correlated to changes in the anthocyanin content, was screened and identified. Under natural ripening conditions, the Z. bungeanum fruit gradually changed in color from green to red, while bagging resulted in the fruit maintaining its green color. After unbagging, the fruit gradually turned red, and the ZbHY5 expression and anthocyanin content increased. In addition, the leaves changed from green to red after exposure to UV-B radiation, and the ZbHY5 expression and anthocyanin content increased. The transient overexpression of ZbHY5 deepened the redness of the Z. bungeanum leaves and promoted the expression of ZbHY5 and ZbMYB113 as well as anthocyanin accumulation. Bimolecular fluorescence complementation (BIFC) showed that there was an interaction between ZbHY5 and ZbMYB113. These results revealed that under UV-B irradiation, ZbHY5 might regulate the expression levels of the structural genes related to anthocyanin biosynthesis through combination with ZbMYB113, thereby affecting anthocyanin accumulation. This finding provides useful insights for further studies focusing on UV-B-induced anthocyanin accumulation in Z. bungeanum .

Zanthoxylum bungeanum is one of the most important medicinal and edible homologous plants because of its potential health benefits and unique flavors. The chemical components in compositions and contents vary with plant genotype variations and various environmental stress conditions. Fatty acids participate in various important metabolic pathways in organisms to resist biotic and abiotic stresses. To determine the variations in metabolic profiling and genotypes, the fatty acid profiling and key differential genes under low temperature stress in two Z. bungeanum varieties, cold-tolerant (FG) and sensitive (FX), were investigated. Twelve main fatty acids were found in two Z. bungeanum varieties under cold stress. Results showed that the contents of total fatty acids and unsaturated fatty acids in FG were higher than those in FX, which made FG more resistant to low temperature. Based on the result of orthogonal partial least squares discriminant analysis, palmitic acid, isostearic acid, linolenic acid and eicosenoic acid were the important differential fatty acids in FG under cold stress, while isomyristic acid, palmitic acid, isostearic acid, stearic acid, oleic acid, linolenic acid and eicosenoic acid were the important differential fatty acids in FX. Furthermore, fatty acid synthesis pathway genes fatty acyl-ACP thioesterase A ( FATA ), Delta (8)-fatty-acid desaturase 2 ( SLD2 ), protein ECERIFERUM 3 ( CER3 ), fatty acid desaturase 3 ( FAD3 ) and fatty acid desaturase 5 ( FAD5 ) played key roles in FG, and SLD2 , FAD5 , 3-oxoacyl-[acyl-carrier-protein] synthase I ( KAS I ), fatty acyl-ACP thioesterase B ( FATB ) and acetyl-CoA carboxylase ( ACC ) were the key genes responding to low temperature in FX. The variation and strategies of fatty acids in two varieties of Z. bungeanum were revealed at the metabolic and molecular level. This work provides a reference for the study of chemical components in plant stress resistance.

Chinese prickly ash (Zanthoxylum) is extensively used as spice and traditional medicine in eastern Asian countries. Recently, an emergent yellow-flower disease (YFD) break out in green Chinese prickly ash (Zanthoxylum schinifolium, Qinghuajiao in Chinese) at Chongqing municipality, and then leads to a sharp reduction in the yield of Qinghuajiao, and thus results in great economic losses for farmers. To address the molecular response for the emergent YFD of Qinghuajiao, we analyzed the transcriptome of 12 samples including the leaves and inflorescences of asymptomatic and symptomatic plants from three different towns at Chongqing by high-throughput RNA-Seq technique. A total of 126,550 genes and 229,643 transcripts were obtained, and 21,054 unigenes were expressed in all 12 samples. There were 56 and 164 different expressed genes (DEGs) for the AL_vs_SL (asymptomatic leaf vs symptomatic leaf) and AF_vs_SF (asymptomatic flower vs symptomatic flower) groups, respectively. The results of KEGG analysis showed that the "phenylpropanoid biosynthesis" pathway that related to plant-pathogen interaction were found in AL_vs_SL and AF_vs_SF groups, and the "Plant-pathogen interaction" found in AF_vs_SF group, implying that this Qinghuajiao YFD might cause by plant pathogen. Interestingly, we detected 33 common unigenes for the 2 groups, and almost these unigenes were up-regulated in the symptomatic plants. Moreover, most of which were homologs to virus RNA, the components of viruses, implying that this YFD was related to virus. Our results provided a primary molecular basis for the prevention and treatment of YFD of Qinghuajiao trees., (© 2021. The Author(s).)

Background and Aims: Zanthoxylum is the only pantropical genus within Rutaceae, with a few species native to temperate eastern Asia and North America. Efforts using Sanger sequencing failed to resolve the backbone phylogeny of Zanthoxylum. In this study, we employed target-enrichment high-throughput sequencing to improve resolution. Gene trees were examined for concordance and sectional classifications of Zanthoxylum were evaluated. Off-target reads were investigated to identify putative single-copy markers for bait refinement, and low-copy markers for evidence of putative hybridization events., Methods: A custom bait set targeting 354 genes, with a median of 321 bp, was designed for Zanthoxylum and applied to 44 Zanthoxylum species and one Tetradium species as the outgroup. Illumina reads were processed via the HybPhyloMaker pipeline. Phylogenetic inferences were conducted using coalescent and maximum likelihood methods based on concatenated datasets. Concordance was assessed using quartet sampling. Additional phylogenetic analyses were performed on putative single and low-copy genes extracted from off-target reads., Key Results: Four major clades are supported within Zanthoxylum: the African clade, the Z. asiaticum clade, the Asian-Pacific-Australian clade and the American-eastern Asian clade. While overall support has improved, regions of conflict are similar to those previously observed. Gene tree discordances indicate a hybridization event in the ancestor of the Hawaiian lineage, and incomplete lineage sorting in the American backbone. Off-target putative single-copy genes largely confirm on-target results, and putative low-copy genes provide additional evidence for hybridization in the Hawaiian lineage. Only two of the five sections of Zanthoxylum are resolved as monophyletic., Conclusions: Target enrichment is suitable for assessing phylogenetic relationships in Zanthoxylum. Our phylogenetic analyses reveal that current sectional classifications need revision. Quartet tree concordance indicates several instances of reticulate evolution. Off-target reads are proven useful to identify additional phylogenetically informative regions for bait refinement or gene tree based approaches., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Stem canker of Zanthoxylum bungeanum is a devastating disease that seriously affects the plantation and industrial development of Z. bungeanum due to a lack of effective control measures. The objective of this study was to screen out resistant Z. bungeanum varieties and further explore their resistance mechanisms against stem canker. Results showed that the most resistant and susceptible varieties were, respectively, Doujiao (DJ) and Fengxian Dahongpao (FD). Combining transcriptomic and metabolomic analyses, we found that the genes and metabolites associated with the phenylpropanoid metabolism, especially flavonoid biosynthesis, were highly significantly enriched in DJ following pathogen infection compared with that in FD, which indicated that the flavonoid metabolism may positively dominate the resistance of Z. bungeanum . This finding was further confirmed by quantitative real-time polymerase chain reaction analysis, through which higher expression levels of core genes involved in flavonoid metabolism in resistant variety were observed. Moreover, by analyzing the differences in the flavonoid content in the stems of resistant and susceptible varieties and the antifungal activities of flavonoids extracted from Z. bungeanum stems, the conclusion that flavonoid metabolism positively regulates the resistance of Z. bungeanum was further supported. Our results not only aid in better understanding the resistance mechanisms of Z. bungeanum against stem canker but also promote the breeding and utilization of resistant varieties.

Background: Apomixis is a form of asexual reproduction that produces offspring without the need for combining male and female gametes, and the offspring have the same genetic makeup as the mother. Therefore, apomixis technology has great application potential in plant breeding. To identify the apomixis types and critical period, embryonic development at different flower development stages of Zanthoxylum bungeanum was observed by cytology., Results: The results show that the S3 stage is the critical period of apomixis, during which the nucellar cells develop into an adventitious primordial embryo. Cytological observations showed that the type of apomixis in Z. bungeanum is sporophytic apomixis. Furthermore, miRNA sequencing, miRNA-target gene interaction, dual luciferase reporter assay, and RT-qPCR verification were used to reveal the dynamic regulation of miRNA-target pairs in Z. bungeanum apomixis. The miRNA sequencing identified 96 mature miRNAs, of which 40 were known and 56 were novel. Additionally, 29 differentially expressed miRNAs were screened according to the miRNAs expression levels at the different developmental stages. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses showed that the target genes of the differentially expressed miRNAs were mainly enriched in plant hormone signal transduction, RNA biosynthetic process, and response to hormone pathways., Conclusions: During the critical period of apomictic embryonic development, miR172c significantly reduces the expression levels of TOE3 and APETALA 2 (AP2) genes, thereby upregulating the expression of the AGAMOUS gene. A molecular regulation model of miRNA-target pairs was constructed based on their interactions and expression patterns to further understand the role of miRNA-target pairs in apomixis. Our data suggest that miR172c may regulates AGAMOUS expression by inhibiting TOE3 in the critical period of apomixis.

Japanese pepper, Zanthoxylum piperitum, is native to Japan and has four well-known lineages (Asakura, Takahara, Budou, and Arima), which are named after their production area or morphology. Restriction-site associated DNA sequencing (RAD-Seq) was used to analyse 93 accessions from various areas, including these four lineages. Single nucleotide variant analysis was used to classify the plants into eight groups: the Asakura and Arima lineages each had two groups, the Takahara and Budou lineages each had one group, and two additional groups were present. In one Asakura group and two Arima groups, the plants were present in agricultural fields and mountains, thus representing the early stage of domestication of the Japanese pepper. The second Asakura lineage group was closely related to plants present in various areas, and this represents the second stage of domestication of this plant because, after early domestication, genetically related lineages with desirable traits spread to the periphery. These results demonstrate that domestication of Japanese pepper is ongoing. In addition, this study shows that spineless plants are polyphyletic, despite the spineless lineage being considered a subspecies of Japanese pepper.

Most chloroplast genes in Zanthoxylum schinifolium (Korean pepper) and Z. piperitum (Japanese pepper) are subject to neutral or purifying selection (Ka/Ks values < 1); however, NAD(P)H dehydrogenase subunit G (ndhG) has a Ka/Ks value of 1.43, which may indicate positive selection. Here, we modeled the ZsNdhG and ZpNdhG structures by comparing them with the NuoJ subunit of respiratory complex I in Escherichia coli, revealing the locations of four amino acid differences between ZsNdhG and ZpNdhG. As these polymorphisms were located at the end of a membrane-spanning α-helix or in peptide loops external to the membrane, they are not expected to have major effects on the membrane-embedding properties of these proteins. However, we found that C-to-U RNA editing occurred at the ndhG-50 sites of ndhG (uCa to uUa, Ser to Leu) in both species, resulting in changes to an amino acid located in the middle of a membrane-spanning α-helix, which may maintain its hydrophobicity. RNA editing at the ndhG-50 site was conserved in many plant species, and the modeled structures of Anthoceros formosae NdhG and Spirodela polyrhiza NdhB provided further evidence that RNA editing increases the hydrophobicity of membrane-embedded proteins. We also speculate that the polar residues inside membrane-spanning α-helices serve to support the protein structure. This report represents the first RNA-editing site identified in Zanthoxylum and points to the importance of considering RNA editing when identifying positively selected genes based on Ka/Ks values., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Zanthoxylum bungeanum, a spice and medicinal plant, is cultivated in many parts of China and some countries in Southeast Asia; however, data on its genome are lacking. In the present study, we performed a whole-genome survey and developed novel genomic-SSR markers of Z. bungeanum. Clean data (∼197.16 Gb) were obtained and assembled into 11185221 scaffolds with an N50 of 183 bp. K-mer analysis revealed that Z. bungeanum has an estimated genome size of 3971.92 Mb, and the GC content, heterozygous rate, and repeat sequence rate are 37.21%, 1.73%, and 86.04%, respectively. These results indicate that the genome of Z. bungeanum is complex. Furthermore, 27153 simple sequence repeat (SSR) loci were identified from 57288 scaffolds with a minimum length > 1 kb. Mononucleotide repeats (19706) were the most abundant type, followed by dinucleotide repeats (5154). The most common motifs were A/T, followed by AT/AT; these SSRs accounted for 71.42% and 11.84% of all repeats, respectively. A total of 21243 non-repeating primer pairs were designed, and 100 were randomly selected and validated by PCR analysis using DNA from 10 Z. bungeanum individuals and 5 Zanthoxylum armatum individuals. Finally, 36 polymorphic SSR markers were developed with polymorphism information content (PIC) values ranging from 0.16 to 0.75. Cluster analysis revealed that Z. bungeanum and Z. armatum could be divided into two major clusters, suggesting that these newly developed SSR markers are useful for genetic diversity and germplasm resource identification in Z. bungeanum and Z. armatum., (© 2020 The Author(s).)

Plants can accumulate a large amount of reactive oxygen species under adverse conditions such as drought and high temperature, which seriously affect the normal growth and development of plants. The antioxidant system can scavenge the reactive oxygen species produced under drought conditions and so mitigate oxidative damage. However, the regulation patterns of many miRNAs under drought stress are still unclear. The content of antioxidant enzymes and the expression patterns of miRNAs and their target genes related to antioxidant systems were studied under drought stress in Zanthoxylum bungeanum. The results indicate that under drought stress, POD, CAT, APX, proline, MDA and related genes all show positive responses to drought, while SOD and its genes showed a negative response. It is indicated that in the antioxidant process of Z. bungeanum, POD, CAT, and APX play a major role, and SOD plays a supporting role. In addition, GUS histochemical and RT-qPCR experimental results show that the expression levels of miRNAs and their target genes are basically negatively correlated, indicating that miRNAs can inhibit the expression of related genes and are also important regulators in the antioxidant system of Z. bungeanum. According to the expression patterns of antioxidant enzymes, miRNA and its target genes under drought stress, combined with previous research results, a model of plant antioxidant mechanism was constructed to provide a reference for further understanding of plant antioxidant mechanism., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Background: Zanthoxylum armatum (Z. armatum) is a highly economically important tree that presents a special numbing taste. However, the underlying regulatory mechanism of the numbing taste remains poorly understood. Thus, the elucidation of the key genes associated with numbing taste biosynthesis pathways is critical for providing genetic information on Z. armatumand the breeding of high-quality germplasms of this species., Results: Here, de novo transcriptome assembly was performed for the five major organs of Z. armatum, including the roots, stems, leaf buds, mature leaves and fruits. A total of 111,318 unigenes were generated with an average length of 1014 bp. Additionally, a large number of SSRs were obtained to improve our understanding of the phylogeny and genetics of Z. armatum. The organ-specific unigenes of the five major samples were screened and annotated via GO and KEGG enrichment analysis. A total of 53 and 34 unigenes that were exclusively upregulated in fruit samples were identified as candidate unigenes for terpenoid biosynthesis or fatty acid biosynthesis, elongation and degradation pathways, respectively. Moreover, 40 days after fertilization (Fr4 stage) could be an important period for the accumulation of terpenoid compounds during the fruit development and maturation of Z. armatum. The Fr4 stage could be a key point at which the first few steps of the fatty acid biosynthesis process are promoted, and the catalysis of subsequent reactions could be significantly induced at 62 days after fertilization (Fr6 stage)., Conclusions: The present study realized de novo transcriptome assembly for the five major organs of Z. armatum. To the best of our knowledge, this study provides the first comprehensive analysis revealing the genes underlying the special numbing taste of Z. armatum. The assembled transcriptome profiles expand the available genetic information on this species and will contribute to gene functional studies, which will aid in the engineering of high-quality cultivars of Z. armatum.

Zanthoxylum bungeanum (Rutaceae), a popular food flavoring and traditional Chinese medicine ingredient, is an important cash crop. Its leaves are rich in flavonoids with multiple bioactivities. However, the transcriptional sequencing has not been investigated, and the molecular basis for the flavonoid biosynthesis remains unclear in this plant. This paper, the key flavonoids (epicatechin, rutin, hyperoside, trifolin, quercitrin, and afzelin) contents were determined in the leaves of 10 Z. bungeanum varieties from a common garden. Results show the leaves of Z. bungeanum mainly contained hyperoside (11.410-21.721 mg/g) and quercitrin (9.401-18.016 mg/g). The total content of these key components was the highest in Fengxian Dahongpao (66.012 mg/g) and the lowest in Fugu (32.223 mg/g). Three varieties (Hancheng stingless, Fugu, and Fengxian Dahongpao) with significant differences in the total content of key flavonoids were selected for transcriptome analysis to obtain flavonoid biosynthesis-related genes. In total, 83 522 unigenes were obtained, 40 668 (48.69%) unigenes were annotated, and 6656 differentially expressed genes (DEGs) were identified. Comparison of the other two varieties, Fugu had many differentially expressed genes indicating the particularity of its variety. Flavonoid-related DEGs of 22 structural genes, including three PALs, one CYP73A, three 4CLs, six CHSs, one CHI, one F3H, one DFR, two ANSs, one ANR, one FLS, and two CYP75B1s, as well as nine MYBs were obtained. These structural genes had different expression patterns in different Z. bungeanum varieties. It is worth noting that the genes expressing the flavonoid 3'5' hydroxylase are absent in Z. bungeanum . Furthermore, quantitative real-time PCR experiment showed consistent results in transcriptome analysis. The RNA-Seq data set of this study sheds lights on the molecular mechanism of flavonoid biosynthesis in Z. bungeanum , provides valuable information for the metabolic regulation of flavonoids, and may serve as a guide for future breeding programs.

High-throughput RNA sequencing has revolutionized transcriptome-based studies of candidate genes, key pathways and gene regulation in non-model organisms. We analyzed full-length cDNA sequences in Zanthoxylum planispinum (Z. planispinum), a medicinal herb in major parts of East Asia. The full-length mRNA derived from tissues of leaf, early fruit and maturing fruit stage were sequenced using PacBio RSII platform to identify isoform transcriptome. We obtained 51,402 unigenes, with average 1781 bp per gene in 82.473 Mb gene lengths. Among 51,402, 3963 unigenes showed variety of isoform. By selection of one representative gene among each of the various isoforms, we finalized 46,306 unique gene set for this herb. We identified 76 cytochrome P450 (CYP450) and related isoforms that are of the wide diversity in the molecular function and biological process. These transcriptome data of Z. planispinum will provide a good resource to study metabolic engineering for the production of valuable medicinal drugs and phytochemicals., (Copyright © 2018. Published by Elsevier Inc.)

Background: To ensure the safety, quality and therapeutic efficacy of processed foods and herbal medicines, it is important to identify and discriminate economically motivated adulterants. Zanthoxylum schinifolium is sold at a higher price than other Zanthoxylum species and is frequently adulterated with closely related Zanthoxylum species because of its high demand as a Korean food ingredient and medicinal material in markets. In addition, the pericarps of three Zanthoxylum species (Z. schinifolium, Z. bungeanum and Z. piperitum) are defined as herbal medicine Zanthoxyli Pericarpium in Korean pharmacopoeias, but not Z. piperitum in Chinese pharmacopoeias. Further confusion arises in the morphological similarity between Z. armatum (adulterant) and Z. bungeanum. Therefore, the aim of this study was to develop a sequence characterized amplified region (SCAR) marker for discrimination of four Zanthoxylum species., Results: With the goal of developing rapid and reliable tools for genetic discrimination of authentic Zanthoxyli Pericarpium, we designed species-specific SCAR markers, based on ITS2 sequences, that generate amplicons of less than 200 bp. Using these markers, we established both conventional and real-time PCR assay methods capable of differentiating samples at the species level. We validated the ability of SCAR markers to authenticate edible oil and herbal medicine, and confirmed that some herbal medicines contaminated with Z. armatum are being distributed as Zanthoxyli Pericarpium in Korean and Chinese markets., Conclusions: The SCAR markers and PCR methods described represent powerful tools for protecting against adulteration and ensuring standardization of processed foods and herbal medicine. © 2018 The Authors. Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2018 The Authors. Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

The WRKY family of transcription factors (TFs) includes a number of transcription-specific groupings that play important roles in plant growth and development and in plant responses to various stresses. To screen for WRKY transcription factors associated with drought stress in Zanthoxylum bungeanum , a total of 38 ZbWRKY were identified and these were then classified and identified with Arabidopsis WRKY. Using bioinformatics analyses based on the structural characteristics of the conservative domain, 38 WRKY transcription factors were identified and categorized into three groups: Groups I, II, and III. Of these, Group II can be divided into four subgroups: subgroups IIb, IIc, IId, and IIe. No ZbWRKY members of subgroup IIa were found in the sequencing data. In addition, 38 ZbWRKY were identified by real-time PCR to determine the behavior of this family of genes under drought stress. Twelve ZbWRKY transcription factors were found to be significantly upregulated under drought stress and these were identified by relative quantification. As predicted by the STRING website, the results show that the WRKYs are involved in four signaling pathways-the jasmonic acid (JA), the salicylic acid (SA), the mitogen-activated protein kinase (MAPK), and the ethylene signaling pathways. ZbWRKY33 is the most intense transcription factor in response to drought stress. We predict that WRKY33 binds directly to the ethylene synthesis precursor gene ACS6 , to promote ethylene synthesis. Ethylene then binds to the ethylene activator release signal to activate a series of downstream genes for cold stress and osmotic responses. The roles of ZbWRKY transcription factors in drought stress rely on a regulatory network center on the JA signaling pathway.

The authentication and traceability of spices is a major concern for industrials and consumers. We focused on species from Zanthoxylum genera which are used for many different applications by local populations and also for trading as spices (dried pericarps or whole fruits). In this case, literature gives contradictory data about botanical names, and commercial labelling is often confusing. We studied commercial fruits pericarps extracts obtained by supercritical CO 2 and analyzed them by GC/MS. The very complex volatile and semi volatile fractions composition of each extract is described. The barcoding method including molecular biology and phylogenetic analyses was also developed in order to check the commercial botanical identification of the raw material. This is a robust method to identify species in berries samples. We used one genetic marker to identify two Rutaceae clusters, including several species of Zanthoxylum genus. These results indicate that Fagara and Zanthoxylum groups could be considered as two different genera. Combination of chemical analysis and DNA analysis provides an original approach to increase chemical and botanical Zanthoxylum genus knowledge., (© 2018 Wiley-VHCA AG, Zurich, Switzerland.)

Fluorescence in situ hybridization (FISH) using oligonucleotide probes for (GAA) 6 (18 bp) and ribosomal DNA (rDNA) (5S rDNA, 41 bp) was applied to analyse Zanthoxylum armatum. (GAA) 6 loci were detected on the pericentromeric regions of five chromosome pairs, and 5S rDNA loci were also detected on the pericentromeric regions of another two chromosome pairs. The densities and locations of (GAA) 6 and 5S rDNA signals varied between individual chromosomes. High-intensity (GAA) 6 signals were detected at the centromeres of two large and two smaller metacentric chromosomes. Relatively strong (GAA) 6 signals were detected at the centromeres of two relatively small metacentric chromosomes, although strong 5S rDNA signals were detected at the centromeres of two additional smaller metacentric chromosomes. Weak (GAA) 6 signals were detected at the centromeres of four large metacentric chromosomes, whereas weak 5S rDNA signals were detected at the centromeres of two smaller metacentric chromosomes. The remaining chromosomes exhibited no signals. Zanthoxylum armatum had 2n = ∼128. The lengths of the mitotic metaphase chromosomes ranged from 1.22 to 2.34 μm. Our results provide information that may be beneficial for future cytogenetic studies and could contribute to the physical assembly of the Zanthoxylum genome.

Real-time reverse transcription quantitative PCR has become a common method for studying gene expression, however, the optimal selection of stable reference genes is a prerequisite for obtaining accurate quantification of transcript abundance. Suitable reference genes for RT-qPCR have not yet been identified for Chinese prickly ash ( Zanthoxylum bungeanum Maxim.). Chinese prickly ash is the source of an important food seasoning in China. In recent years, Chinese prickly ash has also been developed as a medicinal plant. The expression stabilities of ten genes ( 18S , 28S , EF , UBA , UBQ , TIF , NTB , TUA , RPS , and TIF5A ) were evaluated in roots, stems, leaves, flowers and fruits at five developmental stages and also under stress from cold, drought, and salt. To do this we used three different statistical algorithms: geNorm, NormFinder and BestKeeper. Among the genes investigated, UBA and UBQ were found to be most stable for the different cultivars and different tissues examined, UBQ and TIF for fruit developmental stage. Meanwhile, EF and TUA were most stable under cold treatment, EF and UBQ under drought treatment and NTB and RPS under salt treatment. UBA and UBQ for all samples evaluated were most stably expressed, but 18S , TUA and RPS were found to be generally unreliable as reference genes. Our results provide a basis for the future selection of reference genes for biological research with Chinese prickly ash, under a variety of conditions.